Plasminogen (Pg) is an important compound of mammalian blood. On activation by urokinase or streptokinase, the Pg molecule converts to a double chain form called plasmin (Pm). Pm is the natural enzyme that is specific to fibrin, and thus can act to dissolve blood clots in vivo. The structure, modification, and method of functioning of plasminogen/plasmim have been extensively studied. Human plasminogen has been fully sequenced and the positions of the disulfide bonds established. [See Sottrup-Jensen et al. (1978), in Atlas of Protein Sequence and Structure, eds. Dayhoff (National Biomedical Research Foundation, Silver Spring, MD), Vol. 5, suppl. 3, p. 91.]
Human Pg contains 790 amino acids in one polypeptide chain. On activation, the peptide bonds between Arg.sub.560 and Val.sub.561 and Lys.sub.76 and Lys.sub.77 are cleaved. As a consequence plasmin (Lys.sub.77 -Pm) contains two polypeptides, a heavy A chain of 484 amino acids connected by two disulfide bonds to a light B chain of 230 amino acids. Native Pg can be recovered in two glycosylated forms, F-1 and F-2, respectively possessing one or two glycosylation sites: Castellino (1981) (Chemical Reviews), 81:431, at 432-433). The measured molecular weight of human Pg is 92,000 to 94,000. Plasmin acts on Pg to cleave 76 residues between the 76 and 77 position lysines. The trimmed molecule is known as Lys.sub.77 -Pg, or when activated as Lys.sub.77 -Pm. The measured molecular weight of Lys.sub.77 -Pg is from 65,000 to 83,000.
It is the light B chain which contains fibrinolytically active sites, consisting of His.sub.602, Ser.sub.740 and Asp.sub.645. In addition to two disulfide bonds between the A and B chains, plasmin contains at least 20 other disulfide links (Sottrup-Jensen, et al., cited above). The intra-A chain disulfide bonds maintain a special configuration which includes five domains, called "kringles". [See Castellino (1981), cited above, esp. FIG. 4, page 435.]
Because of the molecular size of native plasminogen/plasmin, it has been desired to modify Pm/Pg to shorter chain polypeptides which retain fibrinolytic activity. A smaller molecule fibrinolytic agent can be expected to provide more effective blood clot penetration. One approach to this problem has been to separate the B chain containing the fibrinolytic sites from the A chain. This can be done by disrupting the disulfide bonds between the chains, viz. by a reducing agent like mercaptoethanol. Unfortunately, such reductive separation either disrupts other disulfide bonds or tertiary structure of B chain, drastically reducing fibrinolytic activity of the B chain. [See Christensen, et al. (1979), Biochim. Biophys. Acta, 567:472-481, at 480; Robbins et al. U.S. Pat. No. 4,082,612; and Summaria and Robbins (1976), J. Biol. Chem., 251:5810-5813.] As summarized by Robbins et al., "The plasmin light (B) chain, prepared as described above, has much less proteolytic activity than the plasmin from which it is derived, generally less than about 5% on a molar basis and less than about 15% on a weight basis" (U.S. Pat. No. 4,082,612, col. 3, lines 54-58). The separated B chain is not an effective fibrinolytic agent. If the light chain is complexed with streptokinase, it can be used as an activator of plasminogen, catalyzing the conversion of Pg to Pm. [See Summaria and Robbins (1976), cited above; and Robbins, et al. (1983), Thromb. Haemostas. 50:787-791.]
Enzymatic cleavage of Pg or Pm under conditions where the disulfide bonds are maintained has also been studied. As referred to above, plasmin has an autolysis action on Pg/Pm which cleaves an N-terminal 76 amino acid segment of the A chain. It has been reported with respect to sheep plasminogen that two peptides can be separated by plasmin lysis. The remaining molecule had an estimated molecular weight of 50,000 to 55,000, and was activable to plasmin. [See Paoni and Castellino (1975), Biochem. Biophys. Res. Commun., 65:757-764.] This plasmin lysis of the sheep plasminogen was carried out in a buffered aqueous solution at pH 8.0 for 4 hours at 30.degree. C.
Smaller molecular size activatable plasminogen has been prepared by digestion of Pg with the enzyme elastase. [See Castellino et al. (1981), cited above, at pages 433-434.] A Val.sub.442 -Pg prepared by this procedure was converted to the corresponding active Val.sub.442 -Pm. [Christensen, et al. (1979), Biochim. Biophys. Acta, 567:472-481.] A Leu.sub.449 -Pg was also prepared from porcine plasminogen [Brunisholz and Rickli (1981), Eur. J. Biochem., 119:15.] These molecules had molecular weights of around 38,000, and are apparently among the smallest active Pg/Pm molecules so far reported. However, these polypeptides still contained an A chain segment of more than 200 amino acids including a kringle structure configuration.